Over 150 different types of epithelial cells perform essential vectorial functions required for homeostasis and survival of the organism. A key property underlying these functions is polarity, i.e. the ability of epithelial cells to localize different transporters, channels and hormone receptors to opposite (apical and basolateral) domains of their plasma membrane (PM). The experiments proposed will utilize state of the art molecular, biochemical and microscopic techniques to elucidate molecular sorting mechanisms that regulate the trafficking of molecules essential for epithelial polarity and kidney function/pathobiology. The proposed experiments follow up on our group's recent findings that clathrin plays a broad role of clathrin in basolateral protein sorting and that the ubiquitous (AP-1A) and epithelial-specific (AP-1B) clathrin adaptors carry out complementary basolateral sorting functions in MDCK cells. As we have shown that kidney proximal tubule (KPT) lacks the basolateral sorting adaptor AP-1B, Specific aim 1 will investigate in detail the roles of clathrin adaptors AP-1A, AP-3, AP-4 and GGAs in basolateral sorting in KPT using a domain-selective biotinylation, mass spectroscopy and biochemical and live-imaging trafficking assays. Specific aim 2 will investigate a novel role of clathrin and clathrin adaptors in the apical sorting of Megalin. We have recently described the complete apical recycling pathway of Megalin and our preliminary data suggest that the apical localization of this protein requires clathrin. Specific am 3 will investigate the mechanisms involved in the basolateral sorting of E-cadherin, a fundamental epithelial polarity protein. We will follow up on preliminary data indicating that the basolateral sorting of E-cadherin depends on both AP-1A and AP-1B in the recycling but not in the biosynthetic route, to search for novel TGN and endosomal basolateral sorting mechanisms that depend on sorting signals in the tightly associated protein beta-catenin. Specific aim 4 will characterize the sorting mechanisms of copper transporters A7P7A and ATP7B, highly similar molecules which reside in the TGN in low copper and are released to the PM in low copper. The studies proposed will contribute to a deeper understanding of fundamental epithelial trafficking mechanisms and human (including kidney) diseases associated with malfunction of the molecules under study in this proposal.